Tick-borne encephalitis (TBE) is a viral infection that affects the main parts of the brain and spinal cord of the central nervous system, as well as spinal nerve roots and peripheral nerves. As a result, a TBE infection may result in long-lasting neurological complications, or even death [1
]. There is no specific antiviral medication against TBE infection, and the only currently available treatment for it is restricted to symptomatic therapy. However, TBE can be prevented successfully by vaccination. Currently available commercial vaccines based on the inactivated whole virus show mild and transitory side-effects [2
]. Although this type of vaccine is successful, the manufacturing of inactivated TBE vaccines is associated with processing a large number of dangerous pathogens. Therefore, the development of new vaccines with a safe production process that could cause prolonged immunity is much-needed [3
]. Highly effective and safe vaccines based on the recombinant subunits of viral proteins might represent a promising alternative to the existing vaccines based on the inactivated virus.
Tick-borne encephalitis virus (TBEV) is a spherical virus of the Flavivirus
genus. Virion particles are covered with a glycoprotein coat representing a continuous protein lattice of the homodimers of an envelope protein (protein E) [5
]. The protein E is a 496 residue-long class II fusion protein that plays a key role in the processes of virus particle assembly, virion budding in the endoplasmic reticulum of the host cells, binding of the virus to the cell surface, and fusion of the viral and the host cell membranes. Hence, this protein determines the tropism of the virus. Each monomer of protein E consists of three domains (domains I, II, and III), stem, and a hydrophobic anchor that holds the protein in the lipid membrane of the virion. According to the flavivirus convention, the stem and the hydrophobic anchor form the C-terminal domain IV of the protein E [6
]. Domain III (DIII) is one of three N-terminal domains that form an ectodomain containing about 400 residues, and is located outside the viral membrane. DIII includes the most important epitopes of protein E, which induces antibodies neutralizing the virus and prevents the pH-induced conformational changes of E-proteins required for receptor binding [7
]. However, DIII is not immunogenic due to its low molecular weight (MW 16 kDa). One of the strategies for increasing the immunogenicity of this protein is the creation of chimeric (hybrid) recombinant proteins with specified properties and with decreased DIII toxicity for bacterial host cells [8
]. Heat shock proteins (HSPs) serve as promising fusion partners due to their remarkable effects on the immune system [9
]. However, even large proteins are often weak antigens that need adjuvants. Our previous studies have shown that tubular immunostimulating complexes (TI-complexes) enhance the immune response against different antigens, such as porin OmpF of the enterobacteria Yersinia pseudotuberculosis
], the HA1 subunit of the Influenza A H1N1 hemagglutin (A/California/04/2009(H1N1)) [11
], and the recombinant hemagglutinin monomer of the influenza A virus H1/N1 [12
The nanoparticulate TI-complex is an adjuvanted antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A2
-2 (CDA), and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG forms a lipid matrix for the protein antigen incorporated into TI-complexes. Its fatty acid composition and microviscosity, which depend on the taxonomic position of marine macrophytes, can differently influence the conformation and immunogenicity of a protein antigen [10
The aim of the present work was to express, isolate, and characterize a chimeric HSP70/EIII protein based on the fusion of the bacterial HSP70 of Y. pseudotuberculosis and EIII (DIII + stem) domains of the TBEV E protein as a prospective antigen for the TI-complexes and the development of an anti-TBE subunit vaccine.
3. Results and Discussion
For all constructs analyzed in this study, the commercial vector pET-40b(+) was used. This plasmid has important features, such as the presence of the gene encoding redox protein DsbC, which promotes the correct protein folding of the recombinant protein. It also has an N-terminal signal sequence, which allows the direction of the recombinant protein to the cell periplasm.
To construct three recombinant plasmids using the commercial vector pET-40b(+), two PCR products were created, the amplified 450 base pair (b.p) fragment of the gene encoding domain III of the TBEV E protein and gene dnaK
of 2100 b.p. corresponding to the mature form of HSP70 of Y. pseudotuberculosis.
These two PCR products were inserted between the SacI/SalI and NcoI/SacI-restriction fragments of the pET-40b(+) vector, respectively (Figure 1
To improve the efficiency of ligation, the restricted pET-40b(+) vectors were treated by alkaline phosphatase CmAP for dephosphorylation of plasmid ends to prevent the self-ligation of the vector [34
]. The resultant plasmids 40EIII and 40HSP70 were obtained. To construct chimeric plasmid 40HSP70/EIII encoding a hybrid protein containing mature HSP70 and protein EIII, the plasmid 40HSP70 was digested and the gene encoding for EIII was inserted between the SacI/SalI-restriction fragments of the plasmid. Previously developed expression conditions and the medium MX were used for the expression of all plasmids obtained [14
]. Using the method described in [14
], the soluble forms of HSP70 and HSP/EIII were obtained (Figure 1
An empty plasmid pET-40b(+) was also expressed and a part of the DsbC protein was obtained as a control of the expression and was also used for further purification. The control protein DsbC is a part of all proteins obtained in our study, and is required as a control for other experiments. This protein is a chaperone and a disulfide isomerase. It promotes the correct folding of recombinant proteins in the process of post-translational modification during heterologous expression and also has an E. coli
signal sequence in the N-terminal part that directs the synthesized protein to the periplasmic space of the host cell. After expression of the 40EIII plasmid, the EIII protein overexpression band was not detected in all protein fractions. This observation indicated that the EIII protein, which is toxic to the E. coli
host cells, obviously cannot be expressed separately from other proteins, which probably masks its toxic effects on producing cells. Therefore, we used the EIII protein produced using the continuous exchange cell-free system, as described earlier [8
The conditions for the efficient one-step purification of the expressed proteins were developed. All recombinant proteins analyzed in this study have an N-terminal 6×His-tag that permitted using the metal-affinity chromatography as the most efficient one-step purification. After purification, proteins of 95% purity were obtained according to SDS-PAGE (Figure 2
). The total yields of the purified proteins were 203 mg for HSP70 and 158 mg for HSP70/EIII from 1 L of bacterial culture. The DsbC protein was also purified in small quantities to control its presence/absence in the fractions of HSP70 and HSP70/EIII proteins. According to the SDS-PAGE analysis, the apparent molecular mass values of the purified proteins were 99 kDa for HSP70 (70 kDa HSP70 with the 29 kDa DsbC appendage) and 116 kDa for HSP70/EIII (87 kDa HSP70/EIII with the 29 kDa DsbC appendage).
The mice immunization experiment showed that the individual HSP70/EIII protein induced a two times higher production of anti-EIII antibodies in comparison with the low-molecular EIII protein (16 kDa) alone (Figure 3
). In turn, the incorporation of the chimeric protein in the TI-complex resulted in two-fold increase in the levels of the anti-EIII antibodies.
The use of the Y. pseudotuberculosis
HSP70 as an EIII companion protein likely leads to better antigen presentation [35
] and increases the EIII immunogenicity. This is similar to the HSP70 from Mycobacterium tuberculosis
, which proved its effectiveness as an adjuvant enhancing the immunogenicity of weak antigens of various natures. Proteins fused to HSP70 elicit strong humoral and cellular immune responses [36
]. In addition, HSP70 is able to insert into a lipid membrane [38
] and thereby serves as an anchor of the chimeric HSP70/EIII protein. The binding of HSP70 to lipid membranes depends on the lipid composition [39
] and provides a means for the indirect regulation of the EIII conformation and optimized presentation of DIII in the HSP70/EIII content.
Differential scanning calorimetry (DSC) was used to investigate the integral changes in the biological macromolecules associated with the release or absorption of heat. The extent of the reversibility measured by the relative area recovery seen on the second scan of HSP70 and HSP70/EIII depended on the temperature at which the first scan was terminated before cooling the samples in preparation for the second scan. In all cases, the low-temperature transitions (Figure 4
) were fully reversible. If heating was terminated at temperatures before the second peak maximum, then the second scan showed about 80–90% reversibility, but melting became irreversible when the heating was conducted to higher temperatures. We found, however, that the HSP70 and HSP70/EIII melting results obtained in this work were practically independent of the scan rate, suggesting that the denaturation process was not kinetically determined. Therefore, we concluded that the data could be analyzed semi-quantitatively using thermodynamic models [40
The apparent molar heat capacity of the HSP70, HSP70-EIII, and HSP70-EIII + MGDG samples and the results of the deconvolution conducted by the software provided by MicroCal under the assumption of a two-state model of unfolding are provided in Figure 4
, whereas Table 2
summarizes the thermodynamic data calculated for the individual transitions.
and Table 2
show that thermal denaturation of HSP70 represents a complex process, based on the complex shape of the calorimetric curve containing two clearly visible peaks at 53.8 and 68.2 °C, whereas the deconvolution of this calorimetric curve revealed the presence of three peaks positioned at 54.6, 66.4, and 68.5 °C. It is interesting that the calorimetric curve of a structurally and functionally close HSP90 isolated from porcine brain consisted of two peaks at 53.8 and 63.2 °C, and the presence of only two transitions was further supported by the deconvolution of that calorimetric curve [42
]. Consequently, the third peak at 68.5 °C (Tm
4), found as a result of the deconvolution of the calorimetric curve corresponding to the recombinant HSP70, seems to arise as a result of the DsbC domain melting, which was absent in the HSP90 from the porcine brain.
Although the thermogram of HSP70/EIII contained two heat absorption peaks, the deconvolution of the resulting calorimetric curve into elementary components revealed the presence of four independently melting regions (calorimetric domains), instead of three such domains found in HSP70. Three peaks at 54.2, 67.1, and 70.1 °C corresponded to the melting of HSP70 within the HSP70/EIII chimera. It can be seen that the addition of EIII resulted in some minor stabilization of the chimeric protein, which, in its isolated form, had heat absorption peaks at 54.6, 66.4, and 68.5 °C, respectively. Hence, an additional peak at 58.4 °C (Tm2) likely corresponded to the melting of EIII in the content of HSP70/EIII, since this peak was absent in the melting of the isolated HSP70 construct.
The existence of four independent domains in the HSP70/EIII chimeric protein was further confirmed by the computational analysis of its intrinsic disorder predisposition. Figure 5
represents the results of this analysis and shows that HSP70/EIII has four predominantly ordered regions (residues 20–202, 332–530, 541–769, and 938–1080) that corresponded to the DsbC domain, two independent domains of HSP70, and the EIII domain. In the HSP70/EIII chimeric protein, the DsbC domain, HSP70 protein, and EIII domain are positioned at residues 1–216, 277–912, and 935–1080, respectively. Curiously, artificial linkers introduced to connect constituents of the HSP70/EIII chimeric protein (residues 217–276 and 913–934 between the DsbC domain and HSP70 and between HSP70 and EIII, respectively) were noticeably shorter than the predicted disordered segments in the corresponding regions (residues 203–331 and 770–937), indicating that both the N- and C-terminal tails of HSP70 are significantly disordered.
Presumably, one can use a correlation between the sizes of the ordered regions and the transition enthalpy values to assign observed calorimetric peaks to actual domains of the protein. Based on this hypothesis, it is likely that the first transition with the ΔH1 of 99.5 kcal/mol corresponds to the melting of the ordered core of the N-terminal domain of HSP70 (residues 332–530), the second transition with the ΔH2 of 79.9 kcal/mol corresponds to the melting of the core of the EIII domain (residues 938–1080), and the third transition with the ΔH3 of 116 kcal/mol describes the melting of the ordered core of the C-terminal domain of HSP70 (residues 541–769).
As shown in Figure 4
and Table 2
, all peak maximum temperatures (Tm
4) and therefore the thermal stability of all the HSP70/EIII domains increased in the presence of MGDG from U. lactuca
, which forms a lipid matrix for antigen incorporation into the TI-complex [43
]. Based on the remarkable changes in the Tm
2 and transition enthalpy ΔH
2 values, the glycolipid environment had a particularly strong effect on the EIII conformation.
The deconvolution of the experimental fluorescence spectra of HSP70/EIII into elementary components corresponding to the emission of tryptophan fluorophore [44
] (Figure 6
) revealed the strong effect of U. lactuca
MGDG on the HSP70/EIII tertiary structure. This conclusion follows from the noticeable changes in the contributions of different spectral forms to the total fluorescence (Table 3
This analysis revealed that the main changes occurred in the contents of forms II and III. The spectrum of HSP70/EIII was characterized by the absence of form III, which disappeared as a result of the joining of EIII to HSP70. On the other hand, the addition of glycolipid not only restored this spectral form, but even increased the contribution of form III in the spectrum of HSP70/EIII at the expense of the total disappearance of form II. These rearrangements indicate the existence of a relaxing lipid effect on the protein tertiary structure, which contributes to the “correct” presentation of antigenic determinants and is probably the reason for the significant increase in the production of antibodies against EIII after mice immunization with the HSP70/EIII incorporated in the TI-complex in comparison with the effects of HSP70/EIII alone.